CN116376936B - Method for biosynthesis of ginkgolic acid and gene sequence thereof - Google Patents
Method for biosynthesis of ginkgolic acid and gene sequence thereof Download PDFInfo
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- YXHVCZZLWZYHSA-FPLPWBNLSA-N Ginkgoic acid Chemical group CCCCCC\C=C/CCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-FPLPWBNLSA-N 0.000 title claims abstract description 64
- YXHVCZZLWZYHSA-UHFFFAOYSA-N (Z)-6-[8-pentadecenyl]salicylic acid Natural products CCCCCCC=CCCCCCCCC1=CC=CC(O)=C1C(O)=O YXHVCZZLWZYHSA-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 108090000623 proteins and genes Chemical group 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 34
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 33
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 239000002253 acid Substances 0.000 claims description 17
- 241000196324 Embryophyta Species 0.000 claims description 15
- 244000068988 Glycine max Species 0.000 claims description 15
- 150000007513 acids Chemical class 0.000 claims description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- MBYNDKVOZOAOIS-FPLPWBNLSA-N 2-(10-heptadecenyl)-6-hydroxybenzoic acid Chemical compound CCCCCC\C=C/CCCCCCCCCC1=CC=CC(O)=C1C(O)=O MBYNDKVOZOAOIS-FPLPWBNLSA-N 0.000 claims description 3
- OFFQPVDOVYHTBX-AVQMFFATSA-N 2-[(8E,11E)-heptadeca-8,11-dienyl]-6-hydroxybenzoic acid Chemical compound CCCCC\C=C\C\C=C\CCCCCCCc1cccc(O)c1C(O)=O OFFQPVDOVYHTBX-AVQMFFATSA-N 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 238000000132 electrospray ionisation Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 230000002018 overexpression Effects 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000001819 mass spectrum Methods 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000002137 ultrasound extraction Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 4
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 230000002194 synthesizing effect Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 235000010469 Glycine max Nutrition 0.000 description 14
- 210000004209 hair Anatomy 0.000 description 14
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 241000218628 Ginkgo Species 0.000 description 4
- 235000011201 Ginkgo Nutrition 0.000 description 4
- 235000008100 Ginkgo biloba Nutrition 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 244000194101 Ginkgo biloba Species 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- 108010030975 Polyketide Synthases Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- -1 alkyl phenolic acid Chemical class 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 229930184727 ginkgolide Natural products 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000012499 inoculation medium Substances 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
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Abstract
The invention discloses a method for biosynthesis of ginkgolic acid and a gene sequence thereof, which relate to the technical field of ginkgolic acid synthesis, wherein the nucleotide sequence of a synthesized ginkgolic acid gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; and/or the nucleotide sequence of the synthesized ginkgolic acid gene is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4. The method and the gene sequence for synthesizing the ginkgolic acid by the biosynthesis provide a new target point for synthesizing and regulating the ginkgolic acid and a new thought for high-quality production of ginkgolic acid and related products. In addition, the method is carried out in the organism without adding any other components, so that the ginkgolic acid can be efficiently generated only by normally planting plants transferred with target genes.
Description
Technical Field
The invention relates to the technical field of ginkgolic acid synthesis, in particular to a method for biologically synthesizing ginkgolic acid and a gene sequence thereof.
Background
Ginkgolic acid is a long-chain alkyl phenolic acid metabolite, is produced in the outer seed coats of ginkgo leaves and ginkgo nuts, and is another component with abundant content except flavonoid and ginkgolide in ginkgo. The high content of ginkgolic acid makes the gingko related products have strong toxicity and can cause serious anaphylactic reaction, gene mutation and nerve injury, so that the effective control of the synthesis of ginkgolic acid is one of key factors for improving the germplasm of gingko. In addition, the ginkgolic acid has antibacterial and strong insecticidal effects, can inhibit the growth of tubercle bacillus in vitro, has various pharmacological activities such as anti-tumor, antiviral, antioxidation and the like, and therefore, the high-efficiency synthesis of the ginkgolic acid has good market prospect.
Currently, ginkgolic acids are mainly obtained by chemical synthesis. However, the method has the advantages of high resource requirement, high environmental pollution, complex synthesis process, high difficulty, improper protection in the operation process, and easy generation of vascular irritation symptoms and other series of toxic reactions. Accordingly, biosynthesis is a good approach that can effectively solve the above problems.
In terms of biosynthesis, although the biosynthesis process of ginkgolic acid has been hypothesized and inferred, a key enzyme playing an important role in ginkgolic acid biosynthesis has not been found yet, and heterologous biosynthesis of ginkgolic acid cannot be truly realized.
Disclosure of Invention
The invention aims to provide a method for biosynthesis of ginkgolic acid and a gene sequence thereof, wherein the whole process is carried out in an organism, no other components are needed to be added, and ginkgolic acid can be efficiently generated by only normally planting plants transferred with target genes.
In order to achieve the above purpose, one aspect of the present invention provides a gene for biosynthesis of ginkgolic acid, wherein the nucleotide sequence of the synthesized ginkgolic acid gene is shown as SEQ ID NO.1, and the amino acid sequence of the synthesized ginkgolic acid gene is shown as SEQ ID NO. 2;
and/or the nucleotide sequence of the synthesized ginkgolic acid gene is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
In another aspect, the invention provides a method for biosynthesis of ginkgolic acid, comprising the following steps:
s1, constructing a recombinant vector with the synthesized ginkgolic acid gene;
s2, agrobacterium rhizogenes carrying the recombinant vector constructed in the step S1 is transformed into plants;
s3, culturing the plants obtained in the step S2, and screening positive transformed plants;
s4, extracting the positive plants obtained in the step S3 to obtain ginkgolic acid;
s5, detecting the ginkgolic acid obtained in the step S4.
Preferably, the recombinant vector in step S1 is an over-expression vector with XhoI and XbaI cleavage sites.
Preferably, in step S3, the specific primers for screening positive transformed plants are any of the groups (1) to (2);
(1) The primer pair consists of nucleotide sequences shown as SEQ ID NO.13 and SEQ ID NO. 14;
(2) The primer pair consists of nucleotide sequences shown as SEQ ID NO.15 and SEQ ID NO. 16.
Preferably, in step S4, the method for extracting ginkgolic acid is as follows:
s41, collecting a plant sample, grinding by using liquid nitrogen, and freeze-drying;
s42, adding petroleum ether, performing ultrasonic-assisted extraction, putting into a centrifuge for centrifugation, taking supernatant, adding into a new centrifuge tube, repeating for three times, and combining the three supernatants;
s43, placing the supernatant into a freeze dryer for overnight concentration to obtain the ginkgolic acid dry powder.
Preferably, in step S5, the method and parameters for detecting ginkgolic acid are as follows:
s51, re-dissolving the ginkgolic acid dry powder obtained in the step S43, and filtering the solution by using a 0.22 mu m syringe type filter membrane filter to obtain ginkgolic acid solution;
s52, separating the extracting solution by using high performance liquid chromatography, wherein the specific parameters are as follows: (1) the separation chromatographic column is XBIridge cube BEH C18 (150 mm×3mm,2.5 μm, waters, USA); (2) mobile phase A is water (0.01% formic acid, v/v) and B is methanol; (3) gradient elution procedure: 0min, 10% A,90% B (v/v); stopping analysis for 8min, 100% B, 11min, 100% B, 11.5min, 10% A,90% B,16 min; (4) the flow rate was 0.4ml/min; the column temperature was 35 ℃; the sample was introduced in an amount of 2. Mu.l. (5) An ultraviolet detector (detection wavelength 312 nm);
s53, detecting the extracting solution by using a triple quadrupole mass spectrum of an electrospray ionization system, wherein the specific parameters are as follows: (1) nitrogen temperature: 350 ℃; (2) gas flow rate: 10L/min; (3) spray air pressure: 45psi; (4) capillary voltage: -4000V; (5) the multi-reaction monitoring mode, parent/daughter pair number is as follows: 373/329 (ginkgolic acid 17:1); 371/327 (ginkgolic acid 17:2); 347/303 (ginkgolic acid 15:0); 345/301 (ginkgolic acid 15:1); 319/275 (ginkgolic acid 13:0); the Dwell value is 100; the Fragmentor value is 100; the Collision Energy value is 25; cell accelerator Voltage value is 4; the Polarity value is Negative.
Therefore, the method for synthesizing ginkgolic acid by biological synthesis and the gene sequence thereof have the following technical effects:
(1) The invention provides a novel method for synthesizing ginkgolic acid by green efficient heterologous organisms.
(2) The invention provides a ginkgolic acid major synthesis geneGb_29977、Gb_19155And verify the function thereof, and further disclose the biosynthesis process of both genes.
(3) The invention provides a new target point for synthesizing and regulating ginkgolic acid and provides a new idea for high-quality production of ginkgolic acid and related products thereof.
(4) The invention is carried out in the organism without adding any other components, so that the ginkgolic acid can be efficiently generated only by normally planting the soybeans transferred with the target genes.
(5) The invention reduces the possible poisoning reaction in the synthesis process of ginkgolic acid, belongs to the natural growth process of plants, and reduces the pollution degree to the natural environment in the synthesis process.
Drawings
FIG. 1 is a schematic diagram showing identification of soybean root hair transgene positive seedlings, wherein (a) isGb_19155、Gb_29977、Gb_20355Gel diagram of gene; (b) gel diagram of internal reference actin gene;
FIG. 2 is a schematic diagram of a conventional deviceGb_19155、Gb_29977、Gb_20355Quantitative analysis of gene transcription activity;
FIG. 3 is a schematic representation of the structural formulae of 5 ginkgolic acids in three transgenic soybean root hairs;
FIG. 4 is a schematic diagram of quantitative analysis of 5 ginkgolic acids in three transgenic soybean root hairs;
FIG. 5 is a qualitative schematic of LC-MS/MS of 5 ginkgolic acids in three transgenic soybean root hairs.
Detailed Description
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
1. Constructing a soybean root hair genetic transformation system;
A. construction of the expression vector:
(1) Selecting ginkgo leaves in the month of April growth period to clone and separate genes;
(2) Total RNA was extracted using "SPARKeasy Improved Plant RNA Kit" (Shandong Cisco Biotechnology Co., ltd.);
(3) Reverse transcription into cDNA was performed using "Hifair, iii 1st Strand cDNA Synthesis Kit" (division of Saint Biotech, shanghai Co., ltd.);
(4) The three genes were amplified by PCR using the reagent "2 XTaq Master Mix" (Nanjinouzan Biotechnology Co., ltd.).
Wherein,,Gb_29977the specific primers of the gene fragment are:
forward primer: 5-ATGGGCAGTAACGGAAACATGCAGA-3 (SEQ ID NO. 7);
reverse primer: 5-TCAGACGAGTTTACGGGCAAGAATGC-3 (SEQ ID NO. 8);
Gb_19155the specific primers of the gene are:
forward primer: 5-ATGGCAAGTGCTTCTTCAAGTGCCT-3 (SEQ ID NO. 9);
reverse primer: 5-TCAACCCCATCTCACAATGGCAGAA-3 (SEQ ID NO. 10);
Gb_20355specific primers for (control) genes were:
forward primer: 5-ATGCCGAGTACTGAGACTGATTGCGTTATG-3 (SEQ ID NO. 11);
reverse primer: 5-TTAGACAGAGATTGGGACGCTTCGCAAGAC-3 (SEQ ID NO. 12);
gb_29977 and Gb_19155 are key synthetases for catalyzing alkylphenol (acid) synthesis, namely class III polyketide synthase (PKS III), PKSIII in other species is used as a template, and a CLUSTAL W and neighbor-joining tree method of MEGA6.0 is utilized to construct an evolutionary tree of the PKS III catalytic enzyme superfamily. Wherein DNA sequence information is obtained from NCBI (https:// www.ncbi.nlm.nih.gov).
B. Genetic transformation of soybean root hairs:
(1) Taking several soybean seeds, planting in a greenhouse (photoperiod 16h/8h, illumination intensity 80 μmol×m) −2 *s −1 Temperature 24-26 ℃, humidity 70%);
(2) Will beGb_19155AndGb_29977andGb_20355Inserted between XhoI-XbaI cleavage sites of the over-expression vector, and transformed into Agrobacterium rhizogenes (Agrobacterium rhizogenes) K599-HGCI through ice bath, heat shock and other operations;
wherein,,Gb_29977the nucleotide sequence of (2) is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2;
Gb_19155the nucleotide sequence of (2) is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
Gb_20355The nucleotide sequence of (2) is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6.
(3) Cutting the main root from 7 days old healthy, true leaf developed seedlings with sterile scissors, and chamfering the 0.7-1cm portion of the hypocotyl tip (0.5 cm incision) in the inoculation medium;
(4) Scraping a small part of agrobacterium rhizogenes (Agrobacterium rhizogenes) K599-HGCI which grows and carries a target gene from the oblique incision of the hypocotyl, and then directly putting the agrobacterium rhizogenes into completely wetted sterile vermiculite soil (irrigating with sterile distilled water before use);
(5) After the hypocotyl incision is infected with K559-HGCI, covering with a sterile plastic bag to keep the high humidity of the seedling growing environment, and watering is not needed within 2 weeks after inoculation;
(6) After 2 weeks, gradually opening the tuyere for 2 days to slowly reduce the humidity; the bag was then removed and the plants were checked periodically for moisture and humidity.
(7) After 6 weeks, the soybean roots are pulled out, the roots are cleaned, about 12 root hairs are cut by scissors, and the root hairs are respectively put into a 1.5ml centrifuge tube and frozen at the temperature of minus 80 ℃ until use.
2. Identifying soybean root hairs to synthesize ginkgolic acid;
and A, DNA positive plants are identified:
(1) extracting root hair DNA by using an Edward buffer method;
(2) the soybean root hairs were positively identified using the following primer fragments. The results obtained are shown in FIG. 1. Discovery of35S:: Gb_19155And35S::Gb_20355the positive rate reaches 100 percent,35S::Gb_29977the positive rate reaches 92.31 percent. In FIG. 1, (a) showsGb_19155、Gb_29977、Gb_20355Gel map of gene, (b) gel map of reference actin gene.
Gb_29977The specific primers of the positive plants are:
forward primer: 5-CCAACCACGTCTTCAAAGCA-3 (SEQ ID NO. 13);
reverse primer: 5-TCAGACGAGTTTACGGGCAAGAATGC-3 (SEQ ID NO. 14);
Gb_19155the specific primers of the positive plants are:
forward primer: 5-CCAACCACGTCTTCAAAGCA-3 (SEQ ID NO. 15);
reverse primer: 5-TCAACCCCATCTCACAATGGCAGAA-3 (SEQ ID NO. 16);
Gb_20355specific primers for positive plants (control) were:
forward primer: 5-CCAACCACGTCTTCAAAGCA-3 (SEQ ID NO. 17);
reverse primer: 5-TTAGACAGAGATTGGGACGCTTCGCAAGAC-3 (SEQ ID NO. 18);
B. identification of gene transcription:
(1) total RNA was extracted using "SPARKeasy Improved Plant RNA Kit" (Shandong Cisco Biotechnology Co., ltd.).
(2) This was reverse transcribed into cDNA using Hifair, iii 1st Strand cDNA Synthesis Kit, division of Saint Biotech (Shanghai).
(3) Each gene was repeated three times using fluorescent quantitative PCR analysisGmCons4The gene is used as an internal reference. The results obtained are shown in FIG. 2.
GmCons4Specific primers for fluorescent quantitative PCR analysis were:
forward primer: 5-CGGTGGTTCTATCTTGGCATC-3 (SEQ ID NO. 19);
reverse primer: 5-GTCTTTCGCTTCAATAACCCTA-3 (SEQ ID NO. 20);
Gb_29977specific primers for fluorescent quantitative PCR analysis were:
forward primer: 5-ATCTTCAAACTGGGCCGAGA-3 (SEQ ID NO. 21);
reverse primer: 5-CATAGTCCGCCAAAGTCTGC-3 (SEQ ID NO. 22);
Gb_19155specific primers for fluorescent quantitative PCR analysis were:
forward primer: 5-AAGGTGGTGGGTTCGAGAAT-3 (SEQ ID NO. 23);
reverse primer: 5-GTGCTGCACCGTTCAAGTAA-3 (SEQ ID NO. 24);
Gb_20355specific primers for (control) fluorescent quantitative PCR analysis were:
forward primer: 5-CACAAGCGGAGTAGACATGC-3 (SEQ ID NO. 25);
reverse primer: 5-TGTTGTTCTCGGCCAGATCT-3 (SEQ ID NO. 26);
C. qualitative and quantitative identification of ginkgolic acid
Sample preparation:
(1) collecting soybean root hairs of three transgenic strains, grinding by using liquid nitrogen, and freeze-drying, wherein 1.6g dry weight of sample is weighed for each strain;
(2) adding 5ml petroleum ether, extracting with ultrasonic wave for 30min, centrifuging at 12000rpm in a centrifuge for 15min, collecting supernatant, adding into a new centrifuge tube, repeating for three times, and mixing the three supernatants;
(3) the supernatant was concentrated overnight in a lyophilizer, redissolved with 500. Mu.l of methanol, filtered with a 0.22 μm syringe filter, and the filtered solution was sucked into a liquid bottle and stored at-20deg.C for further use.
Analyzing the extracting solution by using a high performance liquid chromatography mass spectrometer, wherein the specific parameters of the high performance liquid chromatography are as follows: (1) the separation chromatographic column is XBIridge cube BEH C18 (150 mm×3mm,2.5 μm, waters, USA); (2) mobile phase A is water (0.01% formic acid, v/v) and B is methanol; (3) gradient elution procedure: 0min, 10% A,90% B (v/v); stopping analysis for 8min, 100% B, 11min, 100% B, 11.5min, 10% A,90% B,16 min; (4) the flow rate was 0.4ml/min; the column temperature was 35 ℃; the sample was introduced in an amount of 2. Mu.l. (5) Ultraviolet detector (detection wavelength 312 nm). The specific parameters of triple quadrupole mass spectrometry detection using electrospray ionization system were as follows: (1) nitrogen temperature: 350 ℃; (2) gas flow rate: 10L/min; (3) spray air pressure: 45psi; (4) capillary voltage: -4000V; (5) the multi-reaction monitoring mode, parent/daughter pair number is as follows: 373/329 (ginkgolic acid 17:1); 371/327 (ginkgolic acid 17:2); 347/303 (ginkgolic acid 15:0); 345/301 (ginkgolic acid 15:1); 319/275 (ginkgolic acid 13:0); the Dwell value is 100; the Fragmentor value is 100; the Collision Energy value is 25;Cell accelerator Voltage and 4; the Polarity value is Negative. The structural formulas of 5 ginkgolic acids in the root hairs of the three transgenic soybeans are shown in the figure 3, the quantitative analysis of 5 ginkgolic acids is shown in the figure 4, and the figure 5 is a qualitative analysis chart of 5 ginkgolic acids.
Therefore, the invention adopts the method for biosynthesis of ginkgolic acid and the gene sequence thereof, can realize the biosynthesis of ginkgolic acid and obtain key genes for biosynthesis of ginkgolic acidGb_29977AndGb_19155and the soybean root hair genetic transformation system is utilized to carry out heterologous biosynthesis verification.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (6)
1. A gene for biosynthesis of ginkgolic acids, characterized in that: the nucleotide sequence of the synthesized ginkgolic acid gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2;
and/or the nucleotide sequence of the synthesized ginkgolic acid gene is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
2. A method for biosynthesis of ginkgolic acids, which is characterized by comprising the following steps:
s1, constructing a recombinant vector with a synthetic ginkgolic acid gene, wherein the synthetic ginkgolic acid gene is the gene as claimed in claim 1;
s2, transforming the recombinant vector constructed in the step S1 into a soybean plant;
s3, screening a sample carrying a target gene;
s4, extracting ginkgolic acid in the positive sample in the step S3;
s5, detecting the extracted ginkgolic acid.
3. The method for biosynthesis of ginkgolic acids as claimed in claim 2, wherein: the recombinant vector in the step S1 is an over-expression vector with XhoI and XbaI enzyme cutting sites.
4. The method for biosynthesis of ginkgolic acids as claimed in claim 2, wherein: in the step S3, screening the specific primers of the positive transformed plants as any group in a-b;
a. the primer pair consists of nucleotide sequences shown as SEQ ID NO.13 and SEQ ID NO. 14;
b. the primer pair consists of nucleotide sequences shown as SEQ ID NO.15 and SEQ ID NO. 16.
5. The method for biosynthesis of ginkgolic acids as claimed in claim 2, wherein in the step S4, the method for extracting ginkgolic acids is as follows:
s41, collecting a plant sample, grinding by using liquid nitrogen, and freeze-drying;
s42, adding petroleum ether, performing ultrasonic-assisted extraction, putting into a centrifuge for centrifugation, taking supernatant, adding into a new centrifuge tube, repeating for three times, and combining the three supernatants;
s43, placing the supernatant into a freeze dryer for concentration overnight to obtain ginkgolic acid dry powder.
6. The method for biosynthesis of ginkgolic acids of claim 5, wherein in step S5, the method and parameters for detecting ginkgolic acids are as follows:
s51, after redissolving the ginkgolic acid dry powder obtained in the step S43, filtering the solution by using a 0.22 mu m syringe type filter membrane filter to obtain ginkgolic acid solution;
s52, separating the extracting solution by using high performance liquid chromatography, wherein the specific parameters are as follows: (1) the separation chromatographic column is XBIridge BEHC18 with the specification of 150mm multiplied by 3mm and 2.5 mu m, waters, USA; (2) the mobile phase A is water, wherein the mobile phase A contains 0.01 percent of formic acid by volume, and the mobile phase B is methanol; (3) gradient elution procedure: 0min, 10% A,90% B by volume; stopping analysis for 8min, 100% B, 11min, 100% B, 11.5min, 10% A,90% B,16 min; (4) the flow rate was 0.4ml/min; the column temperature was 35 ℃; the sample injection amount is 2 μl; (5) an ultraviolet detector with a detection wavelength of 312nm;
s53, detecting the extracting solution by using a triple quadrupole mass spectrum of an electrospray ionization system, wherein the specific parameters are as follows: (1) nitrogen temperature: 350 ℃; (2) gas flow rate: 10L/min; (3) spray air pressure: 45psi; (4) capillary voltage: -4000V; (5) the multi-reaction monitoring mode, parent/daughter pair number is as follows: 373/329, ginkgolic acid 17:1, a step of; 371/327 ginkgolic acid 17:2;347/303, ginkgolic acid 15:0;345/301, ginkgolic acid 15:1, a step of; 319/275, ginkgolic acid 13:0; the Dwell value is 100; the Fragmentor value is 100; the Collision Energy value is 25; cell accelerator Voltage value is 4; the Polarity value is Negative.
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| CN104770624A (en) * | 2015-02-02 | 2015-07-15 | 天津科技大学 | Application of salicylate decarboxylase in degradation of ginkgoic acid |
| CN114645039A (en) * | 2022-04-26 | 2022-06-21 | 天津科技大学 | Mutant salicylate decarboxylase, strain and application thereof in degradation of ginkgolic acid |
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| CN104770624A (en) * | 2015-02-02 | 2015-07-15 | 天津科技大学 | Application of salicylate decarboxylase in degradation of ginkgoic acid |
| CN114645039A (en) * | 2022-04-26 | 2022-06-21 | 天津科技大学 | Mutant salicylate decarboxylase, strain and application thereof in degradation of ginkgolic acid |
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| Transcriptional and Post-Translational Regulation of Plant bHLH Transcription Factors during the Response to Environmental Stresses;Yasmina Radani et al.;《Plants》;第12卷;第1-21页 * |
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